This invention relates to a technique of enriching decomposing bacteria which can be used to treat soil polluted by organochlorine compounds, such as agricultural chemicals, particularly to prevent contamination of river water and groundwater caused by agricultural chemicals in soil, and a technique of isolating the decomposing bacteria by utilizing the enriching technique, and more particularly to techniques of these kinds for enriching and isolating decomposing bacteria for decomposing organochlorine agricultural chemicals difficult to decompose.
To maintain today""s agricultural production, agricultural chemicals cannot be dispensed with, and to conserve flora in golf courses or the like as well, agricultural chemicals are used in large quantities. On the other hand, there is a concern that agricultural chemicals work as contaminants to have undesirable effects on the environment, especially to be a pollution source of river water and groundwater. An organochlorine agricultural chemical pentachloronitrobenzene (xe2x80x9cPCNBxe2x80x9d) used for killing bacteria causing soil disease is pointed out to be one of contaminants causing such a pollution. PCNB is an organochlorine compound very difficult to decompose, and as matters stand, a method of efficiently disposing PCNB remaining in soil and the like has not been proposed.
To restore soil contaminated by organochlorine compounds, such as agricultural chemicals, with decomposing bacteria has been considered to be a useful technique. This technique makes use of decomposing bacteria, among microbes inhabiting in soil in enormous numbers, which are capable of decomposing organic compounds serving as functional skeletons in agricultural chemicals and the like, thereby rendering the organic compounds harmless or eliminating the same from the environment. Therefore, it is possible to eliminate contaminants, such as agricultural chemicals or the like, from the environment by collecting bacteria which are capable of decomposing organochlorine compounds and exploiting such capabilities of the decomposing bacteria.
The method of selective enrichment/isolation of a specific kind of bacteria from various soil-inhabiting bacteria includes a soil percolation technique in which a column or the like is filled with soil containing inhabiting decomposing bacteria to form an enrichment soil layer, and an inorganic salt medium, which contains only organochlorine compounds, such as agricultural chemicals, as solo carbon and nitrogen sources, is continuously circulated through the enrichment soil layer, whereby a specific kind of decomposing bacteria, that is, decomposing bacteria which is capable of using the carbon or nitrogen source contained in the inorganic salt medium for assimilation or co-metabolism is selectively enriched for isolation. Actually, however, the above conventional soil percolation technique generally takes a long time period of one half to one year to enrich and isolate decomposing bacteria, and depending on the kind of an organic compound, there are cases where no suitable decomposing bacteria can be enriched by the method. The fact that such a long time period is required is a large impediment encountered in putting to practical use the river water or groundwater pollution control technique using decomposing bacteria.
The present invention has been made under these circumstances, and an object thereof is to improve the conventional soil percolation technique to thereby provide a method of enriching and isolating decomposing bacteria capable of decomposing an organochlorine agricultural chemical PCNB which is difficult to decompose, in a short time period, and to provide decomposing bacteria for efficiently disposing of PCNB.
To attain the object, the present inventors have improved the conventional soil percolation technique in the following respects, and thereby established the technique which is capable of largely enhancing the speeds of enrichment and isolation of decomposing bacteria. The gist of the improvement is that a porous material having an infinite number of micropores is fragmented to pieces of approximately several mm to ten and several mm in size such that the porous material can be handled with ease and at the same time has a large effective surface area, and then the fragmented porous material is mixed into an enrichment soil layer as an artificial microhabitat. According to this technique, decomposing bacteria can be effectively enriched and isolated over a time period of approximately three weeks to three months, although the required time period is slightly different depending on the kind of a contaminant and the kind of bacteria decomposing the contaminant. The inventors have already filed a patent application concerning the technique (Japanese Patent Application No. Hei 9-30176).
According to the present invention, by using the improved soil percolation technique, a specific kind of decomposing bacteria were selectively enriched and isolated by continuously circulating an inorganic salt medium containing an organochlorine agricultural chemical PCNB as only sources of carbon and nitrogen to the enrichment soil layer. As a result, aerobic bacteria could be obtained in a very short time period which effectively carries out complete decomposition of PCNB. Out of the enriched and isolated decomposing bacteria, three strains which have a high PCNB-decomposing activity were examined for identification, and it was found that the three strains belong to aerobic bacteria named Burkholderia cepacia. As far as the inventors know, decomposing bacteria for decomposing PCNB are mostly anaerobic bacteria, and Burkholderia cepacia obtained by the improved soil percolation technique (Burkholderia cepacia KTYY97, National Institute of Bioscience and Human Technology Agency of Industrial Science and Technology, 1-3, Higashi 1-chrome, Tsukuba-shi, Ibaraki 305-8566, Japan, Receipt No. FERM BP-6721, Received May 18, 1998, hereinafter referred to as xe2x80x9cthe present decomposing bacteriaxe2x80x9d) provides possibility of quite novel uses since this bacteria are aerobic and completely decomposes or degrades PCNB.
It is found that if a loopfulxe2x80x94using an inoculating needlexe2x80x94of the present decomposing bacteria is added to 30 ml of an inorganic salt medium containing PCNB in a concentration of 3 to 4 mg/liter, it is possible to completely decompose PCNB in approximately five days such that all chlorine atoms bonded to each PCNB molecule are removed from the molecule. Further, the study of the present inventors revealed that the present decomposing bacteria are capable of decomposing even an organochlorine agricultural chemical CNP which has been conventionally considered to be very difficult to decompose. Aerobic bacteria, such as the present decomposing bacteria, are less restricted in the manner of handling the same, unlike anaerobic bacteria, and hence only by applying the present decomposing bacteria to a soil contaminated by PCNB, it is possible to effectively decompose the PCNB, which makes it possible to prevent river water and groundwater pollution.
The present decomposing bacteria can be enriched and isolated by the improved soil percolation technique proposed by the present inventors. This method comprises mixing a soil containing an organochlorine agricultural chemical PCNB with a fragmented porous material having an infinite number of micropores and at the same time a greater adsorptivity for adsorbing PCNB than the soil to form an enrichment soil layer, and circulating through the enrichment soil layer an inorganic salt medium containing a carbon and nitrogen source, the carbon and nitrogen source being formed by only PCNB, thereby enriching the decomposing bacteria in the fragmented porous material.
The soil containing the organochlorine agricultural chemical PCNB is preferably a soil continuously using PCNB as an agricultural chemical. The present decomposing bacteria are hardly populated in ordinary soils, and on the other hand, is relatively thickly populated in a soil using PCNB. Therefore, by making use of such a soil, the present decomposing bacteria can be efficiently enriched.
It is preferred that the fragmented porous material used for the enrichment is formed by fragmenting a porous material having an infinite number of micropores, such as charcoal, to pieces of several to ten and several mm in size, and the fragmented porous material is mixed as an artificial microhabitat into the enrichment soil layer. Further, to carry out speedy enrichment of the present decomposing bacteria, it is preferable to use charcoal of broad-leaved tree which is baked at a low or medium temperature of approximately 400 to 700xc2x0 C., with a volume ratio of micropores with a diameter of approximately 5 to 20 xcexcm to a total of micropores being 10% or more, the inside of micropores being slightly alkaline with pH of approximately 7 to 8, and further with a specific surface area of the material being approximately 80 to 120 m2/g. The results of experiments of enrichment made by using various artificial microhabitats different in micropore distribution, pH within micropores, and specific surface area showed that the artificial microhabitat satisfying the above conditions makes it possible to enrich the present decomposing bacteria at a highest speed and with a highest efficiency. Presumably, this is because the artificial microhabitat satisfying the above conditions is readily inhabited by the present decomposing bacteria and a manner of adsorption of PCNB in the micropores permits the present decomposing bacteria to use PCNB efficiently.
The method of isolating the present decomposing bacteria is carried out by using the above enriching method, and comprises mixing a fragmented porous material having the present decomposing bacteria enriched therein into a new fragmented porous material to form an enrichment layer consisting of the fragmented porous materials only, and circulating through the enrichment layer an inorganic salt medium containing a carbon and nitrogen source, the carbon source and the nitrogen source being formed by only PCNB, thereby effecting enrichment of the present decomposing bacteria in the new fragmented porous material as well to increase a degree of enrichment of the present decomposing bacteria, for isolation thereof. To carry out this isolating operation at a high speed and with efficiency, it is preferred that the artificial microhabitat satisfying the above conditions be used as the new fragmented porous material and the isolating operation be repeatedly carried out.
Further, the fragmented porous material having the present decomposing bacteria enriched therein by the enriching and isolating methods according to the invention can be used as an organochlorine agricultural chemical-decomposing bacteria holdback carrier. Thus, the capability of taking out the present decomposing bacteria in the form of a microhabitat which can be easily handled, i.e. as a carrier holding the present decomposing bacteria facilitates application of the holdback carrier holding the present decomposing bacteria to a soil contaminated by PCNB, e.g. mixing the same into a target soil where PCNB should be disposed of, and makes it possible to decompose PCNB in the soil without providing a special facility.